黄芪活性多糖APS-Ⅱ的酸降解及其降解寡糖的结构和免疫活性研究  

Structure and identification of immune fragments of acid-degraded oligosaccharides from active Astragalus polysaccharides APS-Ⅱ

作　　者：石丽霞 李科[1,2,3,4] 焦思明 崔连杰 秦雪梅 杜昱光[4] 李震宇 李晓霞[5] SHI Li-xia;LI Ke;JIAO Si-ming;CUI Lian-jie;QIN Xue-mei;DU Yu-guang;LI Zhen-yu;LI Xiao-xia(Modern Research Center for Traditional Chinese Medicine of Shanxi University,Taiyuan 030006,China;Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education of Shanxi University,Taiyuan 030006,China;Key Laboratory of Effective Substances Research and Utilization in TCM of Shanxi Province,Taiyuan 030006,China;Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China;Shanxi Fruit Industry Work Station,Taiyuan 030001,China)

机构地区：[1]山西大学中医药现代研究中心,山西太原030006 [2]山西大学化学生物学与分子工程教育部重点实验室,山西太原030006 [3]地产中药功效物质研究与利用山西省重点实验室,山西太原030006 [4]中国科学院过程工程研究所,北京100190 [5]山西省果业工作总站,山西太原030001

出　　处：《药学学报》2021年第8期2266-2275,共10页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81872962);国家博士后科学基金(2019M650851);国家重点研发计划(2019YFC1710800);山西省重点研发计划重点项目(201603D311101);山西省优秀人才科技创新项目(201605D211030,201705D211020).

摘　　要：课题组前期研究发现黄芪免疫活性多糖APS-Ⅱ具有较强的免疫活性,鉴于其分子量较大且结构复杂,本文旨在建立APS-Ⅱ酸降解的最优方法,对降解产物进行结构初探与分离制备以后,通过体外免疫细胞实验筛选免疫活性最强的寡糖活性中心。本研究以APS-Ⅱ为研究对象,采用“自上而下”的研究策略,首先通过单因素试验和正交试验优化了APS-Ⅱ酸降解的最佳条件,接着以最佳条件制备的黄芪寡糖通过亲水作用色谱-电喷雾电离源-高分辨飞行时间质谱进行结构解析,然后将黄芪寡糖通过纯化制备色谱仪分离制备不同聚合度的寡糖片段,最后利用体外免疫细胞从多角度筛选免疫活性中心片段。结果表明APS-Ⅱ的最佳酸解条件为水解温度80℃,三氟乙酸浓度1.0 mol·L-1,水解时间1 h;该降解条件具有良好的重复性;降解产物黄芪寡糖含有主链1→4连接的六碳醛聚糖结构;6个寡糖片段的免疫活性筛选实验表明分子量较大的寡糖具有更强的免疫促进作用,推测黄芪寡糖的免疫活性中心位于聚合度DP9~DP19的糖链中。动物福利和实验过程均遵循山西大学动物伦理委员会的规定。该结果对其他中药寡糖的研究具有一定示范作用,为黄芪寡糖的结构表征及构效关系研究奠定基础,同时可以促进黄芪寡糖药物的开发。We previously reported that active Astragalus polysaccharides APS-Ⅱgenerate strong immune activity.Here we establish the optimal method for APS-II acid degradation.After preliminary structural studies and separation and preparation of the degradation products,the oligosaccharide active center with the strongest immune activity was identified by in vitro immune cell culture experiments.The optimum acid degradation conditions for APS-II were determined by a single factor experiment and an orthogonal experiment.Astragalus oligosaccharides prepared under the optimal conditionswere subjected to structural analysis by hydrophilic interaction chromatography-electrospray ionization source-high resolution time-of-flight mass spectrometry.The products were separated and oligosaccharide fragments with different degrees of polymerization were isolated by preparative purification chromatography.Finally,fragments of the immunologically active centers were identified by in vitro immune cell cultures from multiple perspectives.The results show that the optimal acid hydrolysis conditions for APS-Ⅱare hydrolysis temperature 80℃,trifluoroacetic acid concentration 1.0 mol·L-1,hydrolysis time 1 h.The degradation conditions have good repeatability.The degradation product is a six-carbon aldehyde glycan structure with the main chain 1→4 connected.The immune activity screening experiment for six oligosaccharide fragments showed that larger molecular weight oligosaccharides have stronger immune-promoting effects.It is speculated that the immunologically active center of Astragalus oligosaccharide is located in the sugar chain of DP9-DP19.The animal welfare and the experimental process in this study follow the requirements of the Animal Ethics Committee of Shanxi University.This result suggests a foundation for the structural characterization and structure-activity relationship research of Astragalus oligosaccharides,and may promote the development of Astragalus oligosaccharide drugs.

关 键 词：APS-Ⅱ 部分酸水解 质谱解析 分离制备 免疫活性评价 

分 类 号：R282.5[医药卫生—中药学]